The present proposal addresses the biochemistry and cell biology of Class II MHC-restricted antigen presentation. We shall develop new chemical tools that allow us to track protein antigens and their conversion to Class II MHC peptide complexes in a manner not previously possible. We shall undertake site-specific incorporation of the non-natural amino acid azidohomoalanine into recombinant antigen (ovalbumin) followed by the specific detection of Class II MHC peptide complexes through chemical modification by means of a Staudinger ligation. We shall further visualize the trafficking pathways of Class II MHC molecules and of the cells that express them, by making the use of a knock-in mouse in which all Class II MHC molecules have been tagged with green fluorescent protein (GFP). These animals afford the unique opportunity of imaging antigen presentation in live, primary antigen presenting cells. The Class I1 MHC-GFP mice will be combined with mutations expected to affect trafficking of Class II MHC molecules. Class II MHC-positive cells (dendritic cells, B cells) from these animals will be examined by video microscopy, as such and -after antigen loading- by confrontation with antigen-specific T cells. Intracellular transport of Class II MHC molecules will be compared to that of the tetraspanins CD63 and CD82, which are believed to contribute to the formation of membrane microdomains essential for antigen presentation. The use of Class II MHC-GFP mice will be combined with a strategy in which, through knock-in technology, mice will be created that contain B cells of known specificity (anti-ovalbumin). These B cells retain the capacity to undergo somatic hypermutation and -more importantly- class switching. We can thus address the important role of the BCR in antigen presentation in primary B cells, and also examine in vivo the importance of immune complexes in the phenomenon of cross-presentation by which Class I MHC molecules present peptide derived from protein antigens acquired from the extracellular environment. The role of the proteases involved in Class II MHC restricted antigen presentation will be pursued further by examining the immune response to ovalbumin as a foreign antigen and as a self-antigen. In addition, we shall explore mouse models of autoimmune disease on the context of cathepsin deficiencies: the NOD mouse (diabetes) and the NOD.H2 H4 mouse (autoimmune thyroiditis). We have backcrossed the CatB, CatL and CatS -/- mutations onto the NOD background and will explore the consequences of these cathepsin deficiencies for onset and severity of disease.